Electrophysiological studies have demonstrated that increases in the excitability of amygdala neurons correlate with short-term pain follwoing injury, suggesting that hyperexcitability of neurons in the amygdala plays a critical role in the modulation of pain-related behaviors. A direct causal link between the changes in intrinsic excitability in the amygdala and the time-dependent alterations in affective behaviors following injury, however, has not been established. This project aims at addressing this question directly by performing whole-cell voltage- and current-clamp experiments in visually identified amygdala neurons in acute mouse brain slices obtained from animals in models of inflammatory and neuropathic pain. Behavioral approaches to measure mechanical and thermal hypersensitivity as well as anxiety-like and depression-like behaviors are used in combination with electrophysiological and molecular genetic techniques to define the physiological roles of molecularly distinct subpopulation of cells in the amygdala in the modulation of pain-related behaviors. Parallel western blot and immunohistochemical experiments aim at examining the expression pattern of endogenous molecular signaling proteins in amygdala neurons at different time points following injury. Further experiments aim at evaluating the physiological roles of individual molecules on long-term changes in distinct sensory and affective behaviors following injury. This is the second year of this project and it has been taking place while in the process of continuing setting up a new laboratory. The accomplishments during this year include the establishment and complete validation of all the behavioral models required for the proposed aims as well as the completion of some of the immunohistochemical studies. We have also been able to successfully run preliminary electrophysiology experiments and have been able to establish a mouse colony that includes most of the genetic strains needed for the proposed aims. Jon Macleod (postbac IRTA) optimized and validated many of the antibodies needed for the proposed histological experiments before moving on to medical school. Gary Soroosh (summer intern), Simon Arango (summer intern) and Yonatan Arnold (Special Volunteer) completed some of the immunohistochemical studies proposed and collected preliminary data for additional studies. Both Gary and Simon successfully completed their summer projects and presented a poster with their findings at the 2014 NIH Summer Poster Day.